Gas-phase or pulsed liquid phase automated protein sequence analysis as originally described by Hunkapillar et al., Meth. Enzymol. 91: 399-413 (1983), has become the method of choice for determining primary structures of proteins, protein fragments and other peptides. This method has revolutionized the field of protein sequencing mainly because of the method's reliability, very high sensitivity, and wide applicability, as compared to previously used techniques. Gas phase protein sequencing, although an expensive analytical methodology, is now performed in a very large number of laboratories throughout the world and has become an essential and integral part of every biotechnology research effort. Instruments built according to the specifications of the Hunkapillar et al. method have become the industry-standard for protein sequencing, and are used in the vast majority of molecular biology laboratories. One such instrument manufactured by Applied Biosystems, Inc., Foster City, Calif., is described in U.S. Pat. Nos. 4,837,726 and 4,852,017.
Most protein/peptide sequencers use a chemical process modified from the Edman method for sequentially degrading peptides [Edman, Acta Chem. Scand. 4:283 (1950); Edman and Begg, Eur. J. Biochem. 1:80 (1967)]. The Edman method uses two repetitive chemical stages to, in effect, "clip off" amino acids one-by-one from one end of a peptide chain. In the first stage, phenyl isothiocyanate is coupled, in the presence of an organic base, to the amino-terminal amino acid of the peptide to be degraded. In the second stage, the coupled amino-terminal amino acid is "clipped off" or cleaved from the end of the peptide chain by treating the peptide with a strong organic acid. The cleaved amino acid residue derivative, an anilinothiazolinone (ATZ) derivative, is then collected as a sample for analysis. Successive repetition of these stages provides sequential samples which correspond to the amino acid sequence of the degraded peptide.
The collected samples are then analyzed to determine the amino acid content of the sample, thereby determining the peptide sequence. The ATZ derivative is usually converted to a more stable form, a phenylthiohydantoin (PTH) derivative, for analysis. In typical automated sequencers, such as the Applied Biosystems instrument and those described by Wittmann-Liebold et al., Anal. Biochem. 75:621 (1976) and Hewick et al., J. Biol. Chem. 256:7990 (1981), the PTH derivative is formed in a separate reaction vessel after the ATZ derivative has been collected from the peptide solution by extraction with an organic solvent. The extracted ATZ derivative is treated with an aqueous solution of a strong organic acid to produce the PTH derivative. The individual PTH derivative samples are then collected and saved for chromatographic analysis. In some automatic sequencers, the PTH derivatives are transferred directly to an analysis system which is part of the instrument [see for example the systems described by Machleidt and Hoffner in Methods in Peptide and Protein Sequence Analysis, Birr Ed. pp 35-47 (Elsevier 1980); and Wittman-Liebold and Ashman in Modern Methods in Protein Chemistry, Tschesche ed. pp. 303-27 (deGruyter 1985); Rodriguez, J. Chromatography 350:217 (1985)].
Usually the PTH derivatives are analyzed by high-performance liquid chromotography ("HPLC"). Reverse-phase HPLC ("RP-HPLC") using silica column packings has shown exceptional utility in peptide sequencing because (1) it is currently the only analytical method capable of reliably distinguishing between all PTH derivatives in a single analytical stage and (2) it is sufficiently sensitive to make sequencing of picomolar peptide samples possible.
However, current sequencing technology employing RP-HPLC is not without its problems. One significant drawback of present RP-HPLC methods, including those employed by the Applied Biosystems instrument and other conventional sequencing protocols, is that the presence of the PTH derivative of tryptophan ("PTH-Trp") cannot reliably be analyzed because RP-HPLC does not distinguish PTH-Trp from 1, 3-diphenylurea ("DPU"), a by-product of the sequential degradation process. This precludes unambiguous identification of tryptophan in sequencer cycles involving this amino acid, thus leading to potentially serious errors in protein sequence determination.
Thus there is a need to provide a method for reliable unambiguous determination of tryptophan residues in peptide sequences which employs a modified gradient protocol for reverse-phase HPLC of PTH amino acid derivatives. There is also a need to provide a method for separating DPU from PTH-Trp.